15-oxo,11{62 pgf{hd 2{b {301 and {331

ABSTRACT

Novel methods are disclosed for transforming PGA2 and 15 Beta PGA2 and their acetates, methyl esters, and acetate methyl esters to various prostanoic acids and esters of the PGE2 and PGF2 series. Some of the latter are novel and are useful for the same pharmacological purposes as PGE2 and PGF2 .

United States Patent Pike et al.

lS-OXOJIB PGF- AND Inventors: John E. Pike; William I. Schneider,

both of Kalanmzoo. Mich.

Assignec: The Upjohn Company, Kalamazoo,

Mich.

Filed: Oct. 3, 1973 Appl. No.: 403,093

Related U.S. Application Data Division of Ser. No. 159,478, July 2,I971, Pat. No. 3,772,350, which is a continuation-in-part of Ser. No.

71,390, Sept. II, I970, abandoned.

U.S. Cl. 260/514 D, 260/468 D Int. Cl. C07C 61/36, CO7c 69/74 Field ofSearch 260/468 D, 514 D [45] Jan. 28, 1975 References Cited OTHERPUBLICATIONS Bandy, Ann, NY. Acad. Sci, I80, 76 (I971).

Primary ExaminerRobcrt Gcrstl Alwrney, Agent, or FirmMorris L. Nielsen 5Claims, N0 Drawings CROSS REFERENCE TO RELATED APPLICATIONS This is adivision of application Ser. No. 159,478, filed July 2, 1971 now US.Pat. No. 3,772,350, which is is a continuation-in-part of our co-pendingapplication Ser. No. 71,390, filed Sept. 11, 1970 now abandoned.

DESCRIPTION OF THE INVENTION This invention relates to novelcompositions of matter, to novel methods for preparing them, and tonovel intermediates used in those methods. This invention also relatesto novel methods for preparing known compounds, and to novelintermediates used in those methods.

In particular, the several aspects of this invention relate toderivatives of prostanoic acid which has the following structure andnumbering:

COOH

Some of the derivatives of prostanoic acid are known as prostaglandins.One of those, prostaglandin E (PGE has the following formula:

Another, prostaglandin F Still another, prostaglandin F (PGF has theformula:

In Formulas I to IV and in the formulas recited hereinsolid line. Thealternative configuration for the sidechain hydroxy at Cl5 is known as Ror epi (beta), and is shown when necessary by attachment of saidsidechain hydroxy to Cl5 with a heavy solid line and hydrogen with adotted line, thus ,"ht H OH.

The prostaglandin corresponding to PGE (Formula II) but with the R orepi configuration at Cl5 will be designated l5BPGE See Nature, 212, 38(1966) for discussion of the stereochemistry of the prostaglandins.

These conventions regarding formulas, names, and symbols for derivativesof prostanoic acid apply to the formulas, names, and symbols givenhereinafter in the specification and claims. When reference is madehereinafter to the compounds of Formulas II to IV, by the symbols PGEPGF a or PGF or to the methyl esters of any of those, 15(S)configuration will be intended and by established custom, S" or alphawill not be mentioned in the name or symbol. For all of the othercompounds recited hereinafter, the configuration at Cl5 will beidentified in the name as 15,8 whenever the 15(R) configuration isintended.

Molecules of the known prostaglandins each have several centers ofasymmetry, and can exist in racemic' (optically inactive) form and ineither of the two enantiomeric (optically active) formss, i.e., thedextrorotatory and levorotatory forms. As drawn, Formulas II to IV eachrepresent the particular optically active form of the prostaglandin inwhich is obtained from certain mammalian tissues, for example, sheepvesicular glands, swine lung, or human seminal plasma, or by carbonyland/or double bond reduction of a prostaglandin so obtained. See, forexample, Bergstrom, et al., Pharmacol. Rev. 20, 1 (1968) and referencescited therein.

The several aspects of this invention relate to novel methods forpreparing PGE PGF and PGF B their acetates and methyl esters, and theISB-epimers of those compounds, to novel intermediates used in thosemethods, to novel methods used to make those intermediates, and tocertain novel and pharmacologically useful analogs of PGE PGF a and PGFThe novel and pharmacologically useful PGE PGF; and PGF B analogs ofthis invention have the formulas:

In Formulas V, VI, and VII, R is hydrogen, alkyl of one to 8 carbonatoms, inclusive cycloalkyl of 3 to 10 carbon atoms, inclusive, aralkylof 7 to 12 carbon atoms, inclusive, phenyl, or phenyl substituted withone to 3 chloro or alkyl of one to 4 carbon atoms, inclusive. Alsoencompassed by Formulas V, VI, and VII are pharmacologically acceptablesalts when R, is hydrogen. In Formulas V and VII, Y is H pH In FormulaV, B is H OH In Formula VI, indicates attachment to the ring in alpha orbeta configuration.

It will be observed that each of the novel compounds of Formulas V andVI has a hydroxy group attached to the ll-position in betaconfiguration. In PGE PGF and PGF B and in the compounds of Formula VII,the hydroxy at C-1 1 is attached in alpha configuration.

With regard to Formulas V, VI, and VII, and examples of alkyl of one to8 carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, and isomeric forms thereof. Examples of cycloalkylof 3 to 10 carbon atoms, inclusive, which includes alkylsubstitutedcycloalkyl, are cyclopropyl, 2-methylcyclopropyl,2,2-dimethylcyclopropyl, 2,3-diethylcyclopropyl, 2-butylcyclopropyl,cyclobutyl, 2- methylcyclobutyl, 3-propylcyclobutyl,2,3,4-triethylcyclobutyl, cyclopentyl, 2,2-dimethylcyclopentyl,3-pentylcyclopentyl, 3-tert-butylcyclopentyl, cyclohexyl,4-tert-butylcyclohexyl, 3-isopropylcyclohexyl, 2,2-dimethylcyclohexyl,cyclohepptyl, cyclooctyl, cyclononyl, and cyclodecyl. Examples ofaralkyl of 7 to l2 carbon atoms, inclusive, are benzyl, phenethyl,lphenylethyl, 2-phenylpropyl, 4-phenylbutyl, 3- phenylbutyl, 2-(l-naphthylethyl), and l-(2- naphthylmethyl). Examples of phenylsubstituted by one to 3 chloro or alkyl ofone to 4 carbon atoms,inclusive, are p-chlorophenyl, m-chlorophenyl, ochlorophenyl,2,4-dichlorophenyl, 2,4,6- trichlorophenyl, p-tolyl, m-tolyl, o-tolyl,p-ethylphenyl, p-tert-butylphenyl, 2,5-dimethylphenyl, 4-chloro-2-methylphenyl, and 2,4-dichloro-3-methylphenyl.

The known prostanoic acid derivatives, PGE PGF a and PGF B and theiresters and pharmacologically acceptable salts are extremely potent incausing various biological responses. For that reason, these compoundsare useful for pharmacological purposes. See, for example, Bergstrom, etal., cited above, and references cited therein. A few of thosebiological responses are systemic arterial blood pressure lowering inthe case of the PGE and PGE )3 compounds as measured, for example, inanesthetized (pentobarbital sodium) pentolinium-treated rats withindwelling aortic anad right heart cannulas; pressor activity, similarlymeasured, for the PGF compounds; stimulation of smooth muscle as shown,for example, by tests on strips of guinea pig ileum, rabbit duodenum, orgerbil colon; potentiation of other smooth muscle stimulants;antilipolytic activity as shown by antagonism of epinephine-inducedmobilization of free fatty acids or inhibition of the spontaneousrelease of glycerol from isolated rat fat pads; inhibition of gastricsecretion in the case of the PGE compounds as shown in dogs withsecretion stimulated by food or histamine infusion; activity on thecentral nervous system; controlling spasm and facilitating breathing inasthmatic conditions; decrease of blood platelet adhesiveness as shownby platelet-to-glass adhesiveness, and inhibition of blood plateletaggregation and thrombus formation induced by various physical stimuli,e.g., arterial injury, and various biochemical stimuli, e.g., ADP, ATP,serotonin, thrombin, and collagen; and in the case of the PGE compounds,stimulation of epidermal proliferation and keratinization as shown whenapplied in culture to embryonic chick and rat skin segments.

Because of these biological responses, these known prostaglandins areuseful to study, prevent, control, or alleviate a wide variety ofdiseases and undesirable physiological conditions in birds and mammals,including humans, useful domestic animals, pets, and zoologicalspecimens, and in laboratory animals, for example, mice, rats, rabbits,and monkeys.

For example, these compounds, are especially the PGE compounds, areuseful in mammals, including man, as nasal decongestants. For thispurpose, the compounds are used in a dose range of about l0 p. g. toabout 10 mg. per ml. of a pharmacologically suitable liquid vehicle oras an aerosol spray, both for topical application.

The PGE and PGF a compounds are useful in the treatment of asthma. Forexample, these compounds are useful as bronchodilators or as inhibitorsof mediators, such as SRS-A and Histamine which are released from cellsactivated by an antigen-antibody complex. Thus, these compounds controlspasm and facilitate breathing in conditions such as bronchial asthma,bronchitis, bronchiectasis, pneumonia and emphysema. For these purposes,these compounds are administered in a variety of dosage forms, e.g.,orally in the form of tablets, capsules, or liquids; rectally in theform of tablets, capsules, or liquids; rectally in the form ofsuppositories; parenterally, subcutaneously, or intramuscularly, withintraveneous administration being preferred in emergency situations; byinhalation in the form of aerosols or solutions for ncbulizers; or byinsufflation in the form of powder. Doses in the range of about 0.0] to5 mg. per kg. of body weight are used 1 to 4 times a day, the exact dosedepending on the age, weight, and condition of the patient and on thefrequency and route of administration. For the above use theseprostaglandins can be combined advantageously with other antiasthmaticagents, such as sympathomimetics (isoproterenol, phenylephrine,ephedrine, etc); xanthine derivatives (theophylline and aminophyllin);and corticosteroids (ACTH and predinisolone). Regarding use of thesecompounds see South African Pat. No. 681,055.

The PGE compounds are useful in mammals, including man and certainuseful animals, e.g., dogs and pigs,

to reduce and control excessive gastric secretion, thereby reducing oravoiding gastrointestinal ulcer formation, and accelerating the healingof such ulcers already present in the gastrointestinal tract. For thispurpose, the compounds are injected or infused intravenously,subcutaneously, or intramuscularly in an infusion dose range about 0.1pg. to about 500 pg. per kg. of body weight per minute, or in a totaldaily dose by injection or infusion in the range about 0.1 to about 20mg. per kg. of body weight per day, the exact dose depending on the age,weight, and condition of the patient or animal, and on the frequency androute of administration.

The PGE PGE PGF 3 compounds are useful whenever it is desired to inhibitplatelet aggregation, to reduce the adhesive character of platelets, andto remove or prevent the formation of thrombi in mammals, including man,rabbits, and rats. For example, these compounds are useful in thetreatment and prevention of myocardial infarcts, to treat and preventpostoperative thrombosis, to promote patency of vascular graftsfollowing surgery, and to treat conditions such as atherosclerosis,arteriosclerosis, blood clotting defects due to lipemia, and otherclinical conditions in which the underlying etiology is associates withlipid imbalance or hyperlipidemia. For these purposes, these compoundsare administered systemically, e.g., intravenously, subcutaneously,intramuscularly, and in the form of sterile implants for prolongedaction. For rapid response, especially in emergency situations, theintravenous route of administration is preferred. Doses in the rangeabout 0.005 to about 20 mg. per kg. of body weight per day are used, theexact dose depending on the age, weight, and condition of the patient oranimal, and on the frequency and route of administration.

The PGE PGF a and PGF 3 compounds are especially useful as additives toblood, blood products, blood substitutes, and other fluids which areused in artificial extracorporeal circulation and perfusion of isolatedbody portions, e.g., limbs and organs, whether attached to the originalbody, detached and being preserved or prepared for transplant, orattached to a new body. During these circulations and perfusions,aggregated platelets tend to block the blood vessels and portions of thecirculation apparatus. This blocking is avoided by the presence of thesecompounds. For this purpose, the compound is added gradually or insingle or multiple portions to the circulating blood, to the blood ofthe donor animal, to the perfused body portion, attached or detached, tothe recipient, or to two or all of those at a total steady state dose ofabout 0.001 to mg. per liter of circulating fluid. It is especiallyuseful to use these compounds in laboratory animals, e.g., cats, dogs,rabbits, monkeys, and rats, for these purposes in order to develop newmethods and techniques for organ and limb transplants.

The PGE compounds are extremely potent in causing stimulation of smoothmuscle, and are also highly active in potentiating other known smoothmuscle stimulators, for example, oxytocic agents, e.g., oxytocin, andthe various ergot alkaloids including derivatives and analogs thereof.Therefore PGE for example, is useful in place of or in combination withless than usual amounts of these known smooth muscle stimulators, forexample, to relieve the symptoms of paralytic ileus, or to control orprevent atonic uterine bleeding after abortion or delivery, to aid inexpulsion ofthe placenta,

and during the puerperium. For the latter purpose, the PGE compound isadministered by intravenous infusion immediately after abortion ordelivery at a dose in the range about 0.01 to about pg. per kg. of bodyweight per minute until the desired effect is obtained. Subsequent dosesare given by intravenous subcutaneous, or intramuscular injection orinfusion during puerperium in the range 0.01 to 2 mg. per kg. of bodyweight per day, the exact dose depending on the age, weight, andcondition of the patient or animal.

The PGE and PGF 5 compounds are useful as hypotensive agents to reduceblood pressure in mammals. including man. For this purpose, thecompounds are administered by intravenous infusion at the rate about0.01 to about 50 pg. per kg. of body weight per minute, or in single ormultiple doses of about 25 to 500 pg. per kg. of body weight total perday.

The PGE PGA and PGF 3 compounds also increase the flow of blood in themammalian kidney, thereby increasing volume and electrolyte content ofthe urine. Therefore, these compounds are useful in managing cases ofrenal disfunction, especially those involving blockage of the renalvascular bed. Illustratively, the compounds are useful to alleviate andcorrect cases of edema resulting, for example, from massive surfaceburns, and in the management of shock. For these purposes, the compoundsare preferably first administered by intravenous injection at a dose inthe range 10 to 1000 pg. per kg. of body weight or by intravenousinfusion at a dose in the range 0.1 to 20 pg. per kg. of body weight perminute until the desired effect is obtained. Subsequent doses are givenby intravenous, intramuscular, or subcutaneous injection or infusion inthe range 0.05 to 2 mg. per keg. of body weight per day.

The PGE PGF a and PGF fl compounds are useful in place of oxytocin toinduce labor in pregnant female animals, including man, cows, sheep, andpigs, at or near term, or in pregnant animals with intrauterine death ofthe fetus from about 20 weeks to term. For thise purpose, the compoundis infused intravenously at a dose 0.01 to 50 pg. per kg. of body weightper minute until or near the termination of the second stage of labor,i.e., expulsion of the fetus. These compounds are especially useful whenthe female is one or more weeks post-mature and natural labor has notstarted, or 12 to hours after the membranes have ruptured and naturallabor has not yet started.

The PGF a PGF B and PGE, compounds are useful for controlling thereproductive cycle in ovulating female mammals, including humans andanimals such as monkeys, rats, rabbits, dogs, cattle, and the like. Forthat purpose, PGE or PGF a for example, is administered systemically,e.g., intravenously, subcutaneously, and intravaginally, at a dose levelin the range 0.001 mg. to about 20 mg. per kg. of body weight of thefemale mammal, advantageously during a span of time startingapproximately at the next expected time of menses or just prior to thattime. Additionally, expulsion of an embryo or fetus (abortion) isaccomplished by similar administration of the compound during the firstthird of the normal mammalian gestation period.

As mentioned above,'the PGE compounds are potent antagonists ofepinephrine-induced mobilization of free fatty acids. For this reason,this compound is useful in experimental medicine for both in vitro andin vivo studies in mammals, including man, rabbits, and rats,

intended to lead to the understanding, prevention, sympton alleviation,and cure of diseases involving abnormal lipid mobilization and high freefatty acid levels, e.g., diabetes mellitus, vascular diseases, andhyperthyroidism.

The novel Formula-V, -VI, and -Vll PGE PGF a and PGF B analogs of thisinvention each cause the biological responses described above for PGEPGF, and PGF, ,3 respectively, and each of these novel compounds isaccordingly useful for the abovedescribed corresponding purposes, and isused for those purposes in the same manner as described above.

PGE PGF, and PGF p and their esters and pharmacologically acceptablesalts are all potent in causing multiple biological responses even atlow doses. For example, PGE is extremely potent in causingvasodepression and smooth muscle stimulation, and is also potent as anantilipolytic agent. Moreover, for many applications, these knownprostaglandins have an inconveniently short duration of biologicalactivity. In striking contrast, the novel analogs of Formulas V, VI, andVII are substantially more specific with regard to potency in causingprostaglandin-like biological responses, and have a substantially longerduration of biological activity. Therefore, each of these novelprostaglandin analogs is surprisingly and unexpectedly more useful thanone of the corresponding above-mentioned known prostaglardins for atleast one of the pharmacological purposes because it has a different andnarrower spectrum of biological activity than the known prostaglandins,and therefore is more specific in its activity and causes smaller andfewer undesired side effects than when the known prostaglandin is usedfor the same purpose. Moreover, because of its prolonged activity, fewerand smaller doses of the novel prostaglandin analog can frequently beused to attain the desired result.

The novel Formula-V, -VI, and -VII prostaglandin analogs are used asdescribed above in free acid form in ester form, or in pharmacologicallyacceptable salt form. When the ester form is used, the alkyl esters arepreferred, especially the alkyl esters wherein the alkyl moiety containsone to 4 carbon atoms, inclusive. Of those alkyl, methyl and ethyl areespecially preferred for optimum absorption of the compound by the bodyor experimental animal system.

Pharmacologically acceptable salts of these prostaglandin analogs usefulfor the purposes described above are those with pharmacologicallyacceptable metal cations, ammonium, amine cations, or quaternaryammonium cations.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium, and from the alkaline earthmetals, e.g.,.magnesium and calcium, although cationic forms of othermetals, e.g., aluminum, zinc and iron, are within the scope of thisinvention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triisopropylamine, N-methylhexylamine, decylamine, dodecylamine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, a-phenylethylamine, B-phenylethylamine.ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about l8 carbonatoms. as well as heterocyclic amines, e.g., piperidine, morpholine,pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e.g.,l-methylpiperidine, 4- ethylmorpholine, l-isopropylpyrrolidine, I-methylpyrrolidine, 1,4-dimethylpiperazine, 2-

methylpiperidine, and the like, as well as amines containingwater-solubilizing or hydrophilic groups, e.g., mono-, di-, andtriethanolamine, ethyldiethanolamine, N-butylethanolamine,Z-amino-l-butanol, 2-amino-2- ethyl-1,3-propanediol,2-amino-2-methyl-l-propanol. tris(hydroxymethyl)aminomethane, N-phenylethanolamine, N-(p-tert-amylphenyl)diethanolamine, galactamine,N-methylglucamine, N- methylglucosamine, ephedrine, phenylephrine,epinephrine, procaine, and the like.

Examples of suitable pharmacologically acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

As discussed above, these noval prostaglandin analogs are administeredin various ways for various purposes; e.g., intravenously,intramuscularly, subcutaneously, orally, intravaginally, rectally,buccally, sublingually, topically, and in the form of sterile implantsfor prolonged action.

For intravenous injection or infusion, sterile aqueous isotonicsolutions are preferred. For that purpose, it is preferred because ofincreased water solubility to use the free acid form or thepharmacologically acceptable salt form. For subcutaneous orintramuscular injection. sterile solutions or suspensions of the acid,salt, or ester form in aqueous or non-aqueous media are used. Tablets,capsules, and liquid preparations such as syrups, elixirs, and simplesolutions, with the usual pharmaceutical carriers are used for oral orsublingual administration. For rectal or vaginal administration,suppositories, tampons, ring devices, and preparations adapted tgenerate sprays or foams or to be used for lavage, all prepared as knownin the art, are used. For tissue implants, a sterile table or siliconerubber capsule or other object containing or impregnated with thesubstance is used.

The novel compounds of Formulas, V, Vl, and Vll wherein R, is other thanhydrogen, i.e., the esters wherein R, is alkyl of l to 8 carbon atoms,inclusive, cycloalkyl of3 to 10 carbon atoms, inclusive, aralkyl of 7 to12 carbon atoms, inclusive, phenyl, or phenyl substituted with l to 3chloro or alkyl of l to 4 carbon atoms, inclusive, are prepared from thecorresponding acids of Formulas V, VI, and VII, i.e., wherein R, ishydrogen, by methods known in the art. For example, the alkyl,cycloalkyl, and aralkyl esters are prepared by interaction of said acidswith the appropriate diazohydrocarbon. For example, when diazomethane isused, the methyl esters are produced. Similar use of diazoethane,diazobutane, l-diazo-2-ethylhexane, diazocyclohexane, andphenyldiazomethane, for example, gives the ethyl, butyl, 2-ethylhexyl,cyclohexyl, and benzyl esters, respectively.

Esterification with diazohydrocarbons is carried out by mixing asolution of the diazohydrocarbon in a suitable inert solvent, preferablydiethyl ether, with the acid reactant, advantageously in the same or adifferent inert diluent. After the esterification reaction is complete,the solvent is removed by evaporation, and the ester purified if desiredby conventional methods, pref- 9 erably by chromatography. It ispreferred that contact of the acid reactants with the diazohydrocarbonbe no longer than necessary to effect the desired esterification,preferably about one to about ten minutes, to

avoid undesired molecular changes. Diazohydrocarbons are known in theart or can be prepared by methods known in the art. See, for example,Organic Reactions, John Wiley & Sons, Inc., New York, N.Y., Vol. 8, pp.389-394 (1954).

An alternative method for esterification of the carboxyl moiety of thenovel PGF-type or PGE-type compounds of Formulas V, VI, and VIIcomprises transformation of the free acid to the corresponding silversalt, followed by interaction of that salt with an alkyl iodide.Examples of suitable iodides are methyl iodide, ethyl iodide, butyliodide, isobutyl iodide, tert-butyl iodide, cyclopropyl iodide,cyclopentyl iodide, benzyl iodide, phenethyl iodide, and the like. Thesilver salts are prepared by conventional methods, for example, bydissolving the acid in cold dilute aqueous ammonia, evaporating theexcess ammonia at reduced pressure, and then adding the stoichiometricamount of silver nitrate.

The phenyl and substituted phenyl esters of the Formula-V, -VI,' and-VII compounds are prepared by silylating the acid to protect thehydroxy groups, for example, replacing each -OH with O-Si(CH Doing thatmay also change -COOH to COO-Si-(CH A brief treatment of the silylatedcompound with water will change COOSi-(CH back to CO0H. Procedures forthis silylation are known in the art and are discussed hereinafter.Then, treatment of the silylated compound with oxalyl chloride gives theacid chloride which is reacted with phenol or the appropriatesubstituted phenol to give a silylated phenyl or substituted phenylester. Then the silyl groups, e.g., -0- Si(CI-I are changed back to OHby treatment with dilute acetic acid. Procedures for thesetransformations are known in the art.

The novel Formula-V, -VI, and -VII acids (R, is hy drogen) aretransformed to pharmacologically acceptable salts by neutralization withappropriate amounts of the coresponding inorganic or organic base,examples of which correspond to the cations and amines listed above.These transformations are carried out by a variety of procedures knownin the art to be generally useful for the preparation of inorganic,i.e., metal or ammonium salts, amine acid addition salts, and quaternaryammonium salts. The choice of procedure depends in part upon thesolubility characteristics of the particular salt to be prepared. In thecase of the inorganic salts, it is usually suitable to dissolve the acidin water containing the stoichiometric amount of a hydroxide, carbonate,or bicarbonate corresponding to the inorganic salt desired. For example,such use of sodium hydroxide, sodium carbonate, or sodium bicarbonategives a solution of the sodium salt of the prostanoic acid derivative.Evaporation of the water or addition or a watermiscible solvent ofmoderate polarity, for example, a lower alkanol or a lower alkanone,gives the solid inorganic salt if that form is desired.

To produce an amine salt, the acid is dissolved in a suitable solvent ofeither moderate or low polarity. Ex-

amplcs of the former are ethanol, acetone, and ethyl acetate. Examplesof the latter are diethyl ether and benzene. At least a stoichiometricamount ofthe amine corresponding to the desired cation is then added tothat solution. If the resulting salt does not precipitate,

it is usually obtained in solid form by addition of a miscible diluentof low polarity or by evaporation. If the amine is relatively volatile,any excess can easily be removed by evaporation. It is preferred to usestoichiometric amounts of the less volatile amines.

Salts wherein the cation is quaternary ammonium are produced by mixingthe acid with the stoichiometric amount of the corresponding quaternaryammonium hydroxide in water solution, followed by evaporation of thewater.

The novel compounds of Formulas V, VI, and VII wherein R is hydrogen ormethyl, i.e., the free acids and the methyl esters, and also PGE PGF andPGF B and the methyl esters of those are prepared by novel methods whichare described hereinafter. For those methods, one of the followingstarting materials is used:

I I H 0 H0 R3 0 m fcooRe VI I I Hmla In Formulas VIIa, VIII, and IX, Ris either hydrogen or methyl, and R is hydrogen or acetyl.

It will be observed that the compounds encompassed by Formula Vlla arealso encompassed by VII. Thus, some Formula-VII compounds are usefulboth as intermediates and for pharmacological purposes.

These Formula-Vlla, -VIII, and -IX starting materials are allderivatives of prostanoic acid. The compounds of Formula VIII are knownin the art or are available by methods known in the art. See, forexample, Weinheimer et al., Tetrahedron Letters, No. 59, 5,185 (1969);H.W. Youngken, Jr. (ed), Food-Drugs from the Sea," Proc. MarineTechnology Society, pp. 31 l-3l4 (1969). The Formula-VIII compoundwherein R and R are both hydrogen is designated l5B--PGA alternatively15(R)PGA or I5-epi- -PGA The other compounds encompassed by Formula VIIIare'designated 15B-PGA acetate, I5,BP- CA methyl ester, and I5BPGAacetate methyl ester.

The compounds of Formula VIIa are new in the art and methods forpreparing them are described hereinafter. The Formula-VIIa compoundwherein R and R are both hydrogen is designated ISB-PGE alternativelyl5(R)-PGE or I5-epiPGE The other compounds encompassed by Formula VIIaare designated I5BPGE IS-acetate, 15B-PGE methyl ester, and ISB-PGEacetate methyl ester.

The compounds of Formula IX are known in the art. See, for example,British Specification No. 1,097,533. Novel methods for preparing theseFormula-IX compounds are described hereinafter. The Formula-IX compoundwherein R and R are hydrogen is designated PGA The other compoundsencompassed by Formula IX are designated PGA acetate, PGA methyl ester,and PGA acetate methyl ester.

All of the compounds of Formulas Vlla, VIII, and IX are obtained byextraction from a marine invertebrate. The compounds of Formula Vlla andVIII, i.e., the 15;? compounds, are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma R. The compounds of Formula IX, i.e., the15(8) or alpha compounds, are obtained from colonies of Plexaurahomomalla (Esper), 1792, forma S.

These Plexaura homomalla forms are members of the subclass Octocorallia,order Gorgonacea, suborder Holaxonia, family Plexauridae, genusPlexaura. See, for example, Bayer, The Shallow-Water Octocorallia of theWest Indian Region, Martinus Nijhoff, The Hague (I961). Colonies ofthese Plexaura homomalla forms are abundant on the ocean reefs in thezone from the low-tide line to about 25 fathoms in the tropical andsubtropical regions of the western part of the Atlantic Ocean, fromBermuda to the reefs of Brazil, including the eastern shore reefs ofFlorida, the Caribbean island and mainland reefs, and the Gulf of Mexicoisland and mainland reefs. These colonies are bush-like or smalltree-like in habit, and are readily identified for collection asPlexaura homomalla (Esper), 1,792, by those of ordinary skill in thisart. Forms R and S are distinguished by the methods described inPreparation 1 below.

The colonies of these two forms of Plexaura homomalla are easilyseparated into an outer bark-like cortex and an inner wiry proteinaceousstem or skeleton. Symbiotic algae or Zooxanthellae are also present inthe colonies. Weinheimer et al., cited above, disclose the occurence ofthe Formula-VIII compounds wherein R and R are both hydrogen and whereinR is methyl and R is acetyl in the air dried cortex of Plexaurahomomalla (Esper)."

The choice of isolation or extraction method is determined by theparticular Formula-Vlla, VIII, or -IX compound desired. Maximum yield ofthe Formula- VIII or IX diester is realized by freezing whole orcoarsely cut or chopped fresh Plexaura homomalla colonies within an hourand preferablly sooner after the colonies are removed from the reef. Forsmall scale colections, this freezing is done advantageously bycontacting the colonies or pieces with solid carbon dioxide. For largerscale collections, other suitable freezing methods are known to the art.The frozen colonies or colony pieces should be kept frozen, preferablybelow about 20 C. until the extraction takes place.

The major component of fresh Plexaura homomalla (Esper), 1,792, forma Ris I5BPGA acetate methyl ester, the Formula-VIII compound wherein R ismethyl and R is acetyl. Relatively minor components are the hydroxylmethyl ester, the acetate, and the hydroxy acid of Formula VIII and the[SB-PGE compounds encompassed by Formula Vlla. Of the latter, the BPGEacetate methyl ester (R is methyl and R is acetyl) is the most abundant.The major component of Plexaura homomalla (Esper), 1,792, forma S is PGAacetate methyl ester, the Formula-IX compound wherein R is methyl and Ris acetyl. Relatively minor components are the hydroxy methyl ester, theacetate. and the hydroxy acid of Formula IX, and the PGE compoundscorresponding to Formula Vlla but having the 15(S) configuration.

When the acetate methyl ester compound of Formula Vlla, VIII, or IX (Ris methyl, R is acetyl) is desired as a starting material, a suitablemethod comprises grinding the frozen whole Plexaura homomalla coloniesor colony pieces, advantageously in a hogger to a particle size with thelargest dimension about 5 mm.. and then extracting the resultingparticles with any of the usual organic solvents, preferably one withmoderate to high polarity, e.g., dichloromethane or methanol,advantageously, for 15 to 30 minutes in a high speed mixer. The desiredcompounds are isolated from the extract by evaporation, and thenchromatography of the resulting residue. By this procedure, about 24 g.of l5B--PGA acetate methyl ester, and about I g. each of 15BPGA methylester and l5/3PGE are obtained by dichloromethane extraction of 1,500 g.of frozne Plexaura homomalla (Esper), 1,792, forma R colonies or colonypieces. Similarly, relatively large amounts of PGA acetate methyl esterare obtained from frozen Plexaura homomalla (Esper), 1,792, forma S.

When the l5-hydroxy methyl ester of Formula Vlla, VIII, or IX (R ismethyl, R is hydrogen) is desired as a starting material, a suitablemethod comprises grinding the frozen whole Plexaura homomalla coloniesor colony pieces as above, and then contacting the resulting particleswith a lower alkanol, preferably methanol or ethanol, at 25 C. forseveral days. The solvent is then evaporated and the residuechromatographed to give substantially larger amounts of the hydroxymethyl ester compound relative to the acetate methyl ester compound.When the contact between the Plexaura homomalla particles and thealkanaol is substantially shorter, substantially the same amount andratio of the various Formula-Vlla, VIII, or -IX compounds is obtainedwith the alkanol as with dichloromethane. An alternative method forobtaining these I5-hydroxy methyl esters is described below.

When l5BPGA l5BPGE or PGA (R and R in Formulas VIIa, VIII, and IX areboth hydrogen) are desired as starting materials in the novel processesof this invention, they are prepared from the corresponding methylesters and IS-acetate methyl esters after those have been extracted fromthe Plexaura homomalla colonies or colony pieces as described above. Asuitable method for removing the acetyl group of each of theFormula-Vlla, -VIII, and IX IS-acetate methyl esters comprises mixingthe acetate methyl ester in lower alkanol solution, preferably inmethanol solution, with a strong acid, e.g., perchloric acid, for aboutl5 hours at 25 C. A suitable method for removing the methyl group of anyofthe Formula-Vlla, VIII, and -IX methyl esters is the enzymatichydrolysis described in West Germany Offenlegungschrift No. 1,937,912,reprinted in Farmdoc Complete Specifications, Book No. 14, N0. 6,869R,Week R Mar. 18, 1970.

Another method for obtaining ISBPGA l5B P- 65 or PGA from Plexaurahomomalla colonies or colony pieces comprises freezing the Plexaurahomomalla colonies or colony pieces, preferably at a temperature belowabout 20 C., and then allowing the colonies or colony pieces to thaw andwarm to a temperature in the range 20 to 30 C. The thawed colonies orcolony pieces are then maintained in the range 20 to 30 C. for at least24 hours. After that treatment, substantially none of the Formula-Vlla,-Vlll, and -IX compounds wherein R is methyl and R is acetyl arepresent, the principal Formula-Vlla, -VIII, and -IX compounds presentbeing those wherein R and R are both hydrogen, the minor componentsbeing those wherein R is methyl and R is hydrogen or wherein R ishydrogen and R is acetyl. As before, Formula-Vlla and -VIII compoundsare obtained from colonies of Plexaura homomalla (Esper), 1,792, formaR, and Formula-IX compounds are obtained from colonies of Plexaurahomomalla (Esper), 1,792, forma S.

A preferred procedure for the PGA and PGE type free acids comprisesgrinding the Plexaura homomalla colonies or colony pieces, preferably toa particle size with the largest dimension about 5 mm., and thenmaintaining the mixture in contact with water at a temperature in therange to C. for at least 24 hours. This mixture is filtered, and thefiltrate is extracted with an appropriate water-immiscible solvent,e.g., ethyl acetate. The solid residue is also extracted with anappropriate solvent, e.g., methanol. The two extracts are evaporated,and the total residue is chromatographed to give Formula-Vila and -Vlllof Formula-IX compounds, the principal component in each case being thecompound wherein R and R are both hydrogen.

Since our invention of the novel processes for transforming PGA and15B-PGA and their methyl esters and acetate methyl esters to the variousprostanoic acids and esters disclosed herein, it has now been found thatsmall amounts of the 5,6-trans compounds of PGA and 15BPGA and theirmethyl esters and acetate methyl esters are also obtained from Plexaurahomomalla (Esper), 1,792, forms R and S. These 5,6- Trans compounds areextracted with and accompany the corresponding PGA -type compoundsthrough many of their transformations. For example, PGA containing5,6-trans-PGA yields a mixture of PGE and 5,6-trans-PGE by the processrepresented in Chart E below.

When it is desired, for pharmacological purposes, to prepare the majorproducts of this invention free of 5,6-trans compounds, those 5,6-transcompounds are separated either from the starting reactants or from theproducts. In either case, several methods are available for separatingthe 5,6-trans-PG compounds from the P6 compounds. One method is by meansof a silversaturated ion-exchange resin (for example, see E. A. Emken etal., J. Am. Oil Chemists Soc. 41, 388 (1964) illustrated below inPreparations 5 and 6. The other method is by preferentially forming amercuric acetate adduct of the 5,6-cis compound which is extractableinto polar solvents illustrated below in Preparation 7.

Following the processes discussed herein and the procedures of theExamples below, the.5,6-trans-PG (and -l5BPG compounds are transformedto other 5,6-trans-PG (and l5B--PG compounds, e.g., 5,6-trans-PGA to5,6-trans-PGE 5,6-trans- [SB-PGA acetate methyl ester to5,6-translSB-PGF a acetate methyl ester, and the like.

As mentioned above, the Formula-Vila, -VIII, and -IX compounds arestarting materials for the preparation of PGE,, PGF a and PGF; p themethyl esters of those, and also the novel compounds of Formulas V andVI, and some of the novel compounds of Formula VII. The novel processesusing these starting materials will now be described.

The Formula-VIII and -lX starting materials are both of the PGA-type.According to the novel processes of this invention, those are firsttransformed to corresponding PGE-type compounds. The chemical reactionsinvolved in those transformations are shown generically in Chart A.

In Chart A, R., is hydrogen, methyl. or -Si-(A) wherein A is alkyl of lto 4 carbon atoms. inclusive. aralkyl of 7 to 12 carbon atoms,inclusive, phenyl. or phenyl substituted with l or 2 fluoro, chloro, oralkyl of l to 4 carbon atoms, inclusive; G is llOR wherein R ishydrogen, acetyl, or Si(A) when R is hydrogen or methyl, and R is--Si(A) when R, is -Si-(A) R is hydrogen or methyl; and B is .Ws H OH tfba.

Thus, Formula X in Chart A encompasses the starting materials ofFormulas VIII and IX obtained from Plexaura homomalla, and alsocompounds of the formula:

wherein R is as defined above, and Z is fB-ShlM wherein A is as definedabove. In Formula XI, of Chart A,

CHART A coca, X

(oxidation) B It will be observed in Chart A that the Formula-XII and-Xlll products each encompass four stereoisomeric groups of compounds.lncluded are compounds with the lla,l5(S) configuration of PGE (Formula11, above), compounds with the configuration of lla,l5BPGE (FormulaVlla) as obtained from Plexaura homomalla (Esper), 1,792, forma R, andboth the 15(S)v and 15(R) compounds with the 113 configuration of thenovel Formula-V compounds of this invention wherein Y is i.e., llB-PGEand l1B,l5B-PGE If the Formula- Xll or -Xlll product is to have the15(S) configuration, e.g., PGE or 11BPGE then the Formula-X startingmaterial should have the 15(S) configuration, i.e., G should be If a 153compound of Formula Xll or XIII is desired, e.g., lfiPGE or llB,l5BPGEthen the Formula- X starting material should have the (R) or l5-epiconfiguration, i.e., G should be As described above, Formula -lXstarting materials wherein R is hydrogen or methyl and R is hydrogen,and with the 15(S) configuration are obtained from Plexaura homomalla(Esper), 1,792, forma S. Those same compounds are also produced byreacting the corresponding 15(R) (beta) compound with a hydrocarbyl orhalohydrocarbyl sulfonyl chloride or bromide, preferably a loweralkylsulfonyl chloride or bromide, especially methanesulfonyl chlorideor bromide, or a benzeneor substituted-benzenesulfonyl chloride orbromide, e.g., p-toluenesulfonyl chloride. This reaction is done in thepresence of at least sufficient tertiary amine, e.g., triethylamine, toabsorb the hydrogen chloride or hydrogen bromide by-product, and at alow temperature, preferably in the range -15 to +15 c. The presence ofan inert liquid diluent, e.g., tetrahydrofuran, is helpful to maintain amobile homogenous reaction mixture. At 0 C. and with methanesulfonylchloride, usually 30 to 60 minutes is a sufficient reaction time. Theproduct is hydrolyzed to a mixture of 15(5) (alpha) and l5(R) hydroxycompounds. These are separated by procedures known in the art, and the15(S) product is purified by procedures known in the art, advantageouslyby chromatography on silica gel. This reaction is also used to transform15(S) FormulalX starting materials wherein R is hydrogen or methyl and Ris hydrogen to the corresponding 15(R) compounds. ln each case, amixture of 15(R) product and 15(S) starting material is obtained, thecomponents of which are separated as described above.

Another method of transforming a [SB-PG compound to a PG compound is byconverting it to a mix ture of PG l5-formate and 15BPG l5-formatecompounds, separating the PG l5-formate, and hydrolyzing the PGl5-formate to the desired PG compound (see .l.E. Pike et al., J. Org.Chem. 34, 3,552 (1969)).

The mixture of alpha and beta l5-formates is prepared by maintaining the158 compound, e.g. ISB-P- GE l5BPGA or ISB-PGF a in formic acid bufferedwith an alkali metal formate in the range 10 to 50 C. until asubstantial amount of the 158 compound, e.g., 15B-PGE l5-formate, hasbeen transformed to the PG 15-formate. The mixture of the PG l5-formateand 15B l5-formates thus obtained is then separated by known methods,e.g., by chromatography.

The PG l5-formate can then be hydrolyzed to the desired PG 15-hydroxycompound. The l5B PG 15- formate yields the corresponding [SB-PGlS-hydroxy compound, which is then recycled through the above steps forfurther isomerization to the PG compound if desired.

This procedure is also useful to transform a PG compound to a ISB-PGcompound, by obtaining a mixture of the intermediate PG l5-formate andlSB-PG l5- formate compounds, separating them, and hydrolyzing them tothe respective PG l5-hydroxy and 15BPG l5-hydroxy compounds. In thiscase, the PG compound is recycled for further isomerization to the 158compound.

Referring again to Chart A, the transformation of starting material X toepoxide X1 is carried out by reacting X with any agent known toepoxidize an aB-unsaturated ketone without reacting with isolatedcarboncarbon double bonds, for example see Steroid Reactions, CarlDjerassi, ed., Holden-Day Inc, 1963, p. 593. Especially preferred areaqueous hydrogen peroxide or an organic tertiary hydroperoxide. See, forexample, Organic Peroxides, A. V. Tobolsky, et al., lntersciencePublishers, N.Y., 1954. For this purpose, the peroxide or hydroperoxideis employed in an amount of at least one equivalent per mole ofFormula-X reactant in the presence of a strong base, e.g., an alkalimetal hydroxide, a metal alkoxide, or a quaternary ammonium hydroxide.For example, there is employed lithium hydroxde, sodium hydroxide,potassium hydroxide, lithium ethoxide, lithium octyloxide, magnesiummethoxide, magnesium isopropoxide, benzyltrimethylammonium hydroxide,tetraethylammonium hydroxide, butyltrimethylammonium hydroxide,butyldiethylphenylammonium hydroxide, benzylethyldimethylammoniumhydroxide, benzyldimethyloctadecylammonium hydroxide,benzyldodecyldimethylammonium hydroxide, decyldimethylphenylammoniumhydroxide, and the like. See, for example, Sidgwick, Organic Chemistryof Nitrogen, Third Edition, rev. by Miller and Springall, Oxford, 1966,pp. 116-127.

The ratio of alpha to beta epoxide formed in the reaction is related tofour factors: the epoxidizing agent, the base, the diluent, and thetemperature. Hydrogen peroxide is employed in the concentrations usuallyavailable, for example 3 to 90 percent, although 30 percent isespecially convenient. When the alpha epoxide is the desired product,tert-butyl hydroperoxide is especially preferred as the epoxidizingagent. Examples of other organic tertiary hydroperoxides useful for thispurpose are tert-pentyl hydroperoxide, decahydronaphthyl hydroperoxide,a,a-dimethylbenzyl hydroperoxide, and l,l-diphenylethyl hydroperoxide.The base is present in the proportion of 0.1-3.0, preferably about0.1-0.5 equivalent of base per mole of starting material X when R ismethyl and R is acetyl; preferably about 1.5-2.5 equivalent of base permole of starting material VIll or IX wherein R and R are hydrogen. Whenthe alpha epoxide is the desired product, lithium hydroxide, lithium ormagnesium alkoxides of l to 8 carbon atoms, and benzyltrimethylammoniumhydroxide are the preferred bases, although the lithium and magnesiumcompounds are especially preferred.

It is advantageous to use an inert liquid diluent in the epoxidationstep to produce a mobile homogenous reaction mixture, for example, alower alkanol, dioxane, tetrahydrofuran, dimethoxyethane,dimethylsulfoxide, or dimethylsulfone. When the alpha epoxide ispreferred, tetrahydrofuran or the less polar dimethoxyethane areespecially preferred as the diluent. A reaction temperature in the range60 to 0 C. is generally preferred, especially below l0 C. The lowertemperatures below -30 C. are especially preferred for favor ingformation of alpha epoxide over beta epoxide. At a temperature of 20 C.,the epoxidation is usually complete in 3 to 6 hours. It is alsopreferred that the reaction be carried out in an atmosphere of an inertgas, e.g., nitrogen, helium, or argon. When the reaction is complete asshown by the absence of starting material on TLC plates (3 percentacetone in dichloromethane), the reaction mixture is neutralized, andthe epoxy product is isolated by procedures known in the art, forexample, evaporation of the diluent and extraction of the residue withan appropriate watenimmiscible solvent, e.g., ethyl acetate.

This transformation of X to XI usually produces a mixture of Formula-XIalpha and beta epoxides both with either the (R) or l5(S) configurationdepending on the configuration at C-l5 in the Formula-X startingmaterial. Although these mixtures are separated into the individualalpha and beta isomers, for example, by chromatography by proceduresknown to be useful for separating alpha and beta epoxide mixtures, it isusually advantageous to transform the Formula-XI mixture of alpha andbeta epoxides to the corresponding mixture of Formula-XII [la and H6hydroxy compounds. The latter mixture is then readily separated into theHot and "B compounds for example, by chromatography of silica gel.

During the transformation of epoxides Xl to alcohols Xll, an alphaepoxide yields an Ila-hydroxy compound, and a beta epoxide yields an[Iii-hydroxy compound. The ratio of alpha to beta epoxides in X], andhence the eventual ratio of Ila and [IB alcohols in Xll, produced fromstarting material X is dependent in large measure on the nature of R inX. Recall that G is defined as wherein R is hydrogen, acetyl, orSi-(A);, wherein A is as defined above. For either definition of G,i.e., R configuration or S configuration, when R is hydrogen, moreFormula-XI beta epoxide is formed than when R is acetyl, and moreFormula-Xl beta epoxide is formed when R is acetyl than when R is Si(AFor example, when G in formula X is the preferred basic hydrogenperoxide epoxidation gives about equal amounts of alpha and betaepoxides, but when G in formula X is H ococua,

about 3 parts of alpha epoxide and one part of beta epoxide areobtained, and when G in formula X is l l wast-(caste,

about 4 parts of alpha epoxide and one part of beta epoxide areobtained, both reactions with the same epoxidation reagent. When G informula X is ti 0H,

about 1 part of alpha epoxide and 3 parts of beta epoxide are obtainedwith basic hydrogen peroxide, but when G is if b-s l (CH3 about 6 partsof alpha epoxide and 4 parts of beta epoxide are obtained with the sameepoxidation reagent.

Each of the novel Formula V and -VI compounds of this invention has ahydroxy attached in beta configuration to the cyclopentane ring. Some ofthe compounds of Formula V, i.e., when Y is I ll OH H bH.

On the other hand, when a prostanoic acid product with the natural Ilaconfiguration for the hydroxy at C-ll is the desired final product,e.g., PGE PGF a or PGF 2 3 there is advantage in choosing a Formula- Xstarting material which gives a greater amount of the alpha epoxideduring the transformation of X to XI. Those would be Formula-X compoundswherein X is H OCOCHs or the corresponding 15(8) compounds.

As mentioned above, the starting materials of Formula X encompass notonly the Formula-VIII and -IX compounds obtained from Plexaura homomallabut also the silyl compounds of Formula X When desired as reactants,these silyl compounds are prepared by silylation of PGA l5BPGA or themethyl esters of those. These silylations are carried out by proceduresknown in the art. See, for example, Pierce, Silylation of OrganicCompounds," Pierce Chemical Co., Rockford, Ill. (1968). The C-l5 hydroxygroup of PGA l5BPGA or their methyl esters is transformed to an O-Si(A)moiety wherein A is as defined above, sufficient silylating agent beingused according to known procedures to accomplish that. The necessarysilylating agents for this purpose are known in the art or are preparedby methods known in the art. See, for example, Post, Silicones and OtherOrganic Silicon Compounds, Reinhold Publishing Corp., New York, NY.(1949). In the case of PGA and l5B-PGA excess silylating agent andprolonged treatment also transform the COOI-I to -COOSi(A) It isoptional in transforming X to XI whether or not this COOH of PGA orl5B-PGA is esterfied to COOSi(A) since that ester group is transformedto COOH during formation and isolation of the Formula-XI epoxideproduct.

The various As of a -Si(A) are alike or different. For example, an--Si-(A) can be trimethylsilyl, dimethylphenylsilyl, ormethylphenylbenzylsilyl.

When it is desired to retain the Si(A) moiety at C-l5 in the Formula-XIepoxide product, for example, to give steric control in a subsequentreaction, it is important in isolating the epoxide that the presence ofacid be avoided and that contact with water be minimized unless thewater is kept cold, i.e., below about C.

Referring again to Chart A, the transformation of epoxide XI to hydroxycompound XII is accomplished by reduction with chromium (II) salts,e.g., chromium (Il) chloride or chromium (ll) acetate. Those salts areprepared by methods known in the art, e.g., Inorganic Syntheses, Vlll,125 (I966); ibid., VI, 144 (1960); ibid. III, 148 (1950); ibid. l, 122(1939); and references cited in those. This reduction is carried out byprocedures known in the art for using chromium (II) salts to reduceepoxides of aB-unsaturated ketones to B-hydroxy ketones. See, forexample, Cole, et al., J. Org. Chem. 19, 131 (1954), and Neher et al.,l-lelv. Chem. Acta 42, 132 (1959). In these reactions, the absence ofair and strong acids is desirable. If it is desired to maintain a Si-(A)moiety on C-l 5, a neutral reaction mixture is preferred. An especiallypreferred procedure is to generate the chromium (II) ion in the presenceof the Formula-XI epoxide, for example, by mixing the epoxide with achromium (III) salt. e.g., the chloride, with metallic zinc in thepresence of acetic acid. The desired (Formula-XII compound is isolatedfrom the reduction reaction mixture by methods known in the art. carebeing taken to minimize contact of the product with acid and water,especially warm water, when retention of a Si(A) at C-lS is desired.

Unexpectedly, amalgamated aluminum metal has also been found to beuseful as a reducing agent in place of chromium (II) salts to transformFormula XI epoxides to Formula XII hydroxy compounds. This reagent waspreviously not known to be useful for this type of reaction. This use ofamalgamated aluminum represents a distinct and separate aspect of thisinvention.

Amalgamated aluminum is prepared by procedures known in the art, forexample, by contacting aluminum metal in the form of foil, thin sheet,turnings, or granules with a mercury (II) salt, for example, mercuricchloride, advantageously in the presence of sufficient water to dissolvethe mercury (II) salt. Preferably, the surface of the aluminum metal isfree of oxide. That is readily accomplished by physical removal of theusual oxide later, e.g., by abrasion or scraping, or chemically, e.g.,by etching with aqueous sodium hydroxide solution. It is only necessarythat the aluminum surface be amalgamated. The amalgamated aluminumshould be freshly prepared, and maintained in the absence of air andmoisture until used.

The reductive opening of the Formula-XI epoxide ring is accomplished bycontacting said epoxide with the amalgamated aluminum in the presence ofa hydroxylic solvent and sufficient inert organic liquid diluent to givea mobile and homogeneous reaction mixture with respect to the hydroxylicsolvent and said epoxide. Among hydroxylic solvents, water is especiallypreferred although lower alkanols, e.g., methanol and ethanols are alsooperable.

Examples of inert organic liquid diluents are normally liquid etherssuch as diethyl ether, tetrahydrofuran, dimethoxyethane, diglyme(dimethyl ether of diethylene glycol), and the like. Especiallypreferred is tetrahydrofuran. When a water-immiscible liquid diluent isused, a mixture of water and methanol or ethanol is especially useful inthis reaction since the latter two solvents also aid in forming thedesired homogeneous reaction mixture. For example, a mixture of diethylether and water is used with sufficient methanol to give a homogeneousreaction mixture.

This reductive opening requires two hydrogen atoms per molecule ofepoxide. Amalgamated aluminum reacts readily with water and more slowlywith other hydroxylic solvents to give hydrogen. One atomic equivalentof aluminum required three molecular equivalents of the hydroxylicsolvent to give three atomic equivalents of hydrogen. Therefore, onemolecular equivalent of epoxide requires two-thirds atomix equivalent ofaluminum and two molecular equivalents of the hydroxylic solvent.Evolution of hydrogen gas (H molecules) is observed during thisreductive opening of the epoxide. It is not known whether the reductiveopening is caused by hydrogen atoms or hydrogen molecules. However, someof the hydrogen gas escapes from the reaction mixture. Therefore, it ispreferred to use an excess of amalgamated aluminum and hydroxylicsolvent, preferably at least one atomic equivalent of aluminum and threemolecular equivalents of hydroxylic solvent per molecular equivalent ofepoxide. Because of the relatively high economic value of the epoxidecompared with amalgamated aluminum and hydroxylic solvents, it ispreferred to assure maximum yields of the desired Formula-XII hydroxycompound by use of substantially greater excess of amalgamated aluminumand hydroxylic solvent, e.g., up to times or more of those reagents thanis theoretically required.

The reductive opening of the epoxide is carried out by mixing a solutionof the epoxide in the organic diluent with the amalgamated aluminum andthe hydroxylic solvent. Since the reaction is exothermic, it is usuallyadvantageous to cool the solution to a low temperature, e.g., to 0C.,before adding the amalgamated aluminum and hydroxylic solvent and tomaintain the reaction mixture in the range 20 to 30 C. by externalcooling. This is especially advantageous when water is used as thehydroxylic solvent. Higher reaction temperatures are operable but notpreferred when a high yield of the Formula-XII products is desired.Stirring is preferred during the reaction since the reaction mixture isheterogeneous with respect to the solution and the amalgamated aluminum.

For reasons not understood, better yields and a shorter reaction timeare usually observed when only part of the amalgamated aluminum is addedat the start of the reaction, additional portions being added during thereaction, e.g., at l-hour intervals, than when the entire amount ofamalgamated aluminum is added at the start of the reaction. A suitableprocedure is to add about one-third of the amalgamated aluminum at thestart, about one-third after one hour, and another third after a secondhour. The course of the reaction is advantageously determined bywithdrawing small portions of the solution and determining the presenceor absence of starting material by thin layer chromatography. Forexample, when R is methyl and G is in Formulas XII and XII, a suitableTLC system is ethyl acetate-cyclohexaneacetic acid (40/60/2), theFormula-XI starting material having r, 0.64, and the two Formula-XIIproducts having r ,O.25 (11B) and r,0.20 (Ila).

As a modification of the above-described process for reductive openingof the epoxide, it has been found quite unexpectedly that instead ofemploying a Formula-XI compound wherein R is hydrogen, the reductiveopening reaction proceeds more smoothly and completely if there is used,instead, an epoxide of the formula wherein G and are as defined aboveand R is a cation of an alkali or alkaline earth metal or a quaternaryammonium group.

Thus, the Formula-XI epoxide compound is treated with a hydroxide oroxide of lithium. sodium, potassium, magnesium, calcium, barium, orstrontium prior to contacting with the aluminum amalgam. Optionally, thequaternary ammonium bases are used for this neutralization, for examplebenzyltrimethylammonium hydroxide. The base is used in equivalent amountto the acid so that R is replaced by the corresponding metal orquaternary ammonium cation. Alternatively, instead of the hydroxides oroxides, there are employed the hydrides, the carbonate, thebicarbonates, or the alkoxides, for example lithium hydride, potassiumcarbonate, sodium bicarbonate, magnesium methoxide, and the like, whichform the corresponding Formula-Xla salts with the Formula-XI free acid.Alternatively, a metal or quaternary-ammonium carboxylate compound orFormula-Xla salt carried forward from the epoxidation step, whetherisolated in that step or not, is employed in the reductive step withaluminum amalgam. It is preferred that the Formula-Xla salt be solublein the organic diluent-alkanol-water or organic diluentwater medium usedfor the reduction step. By using the above-described salts, thereduction step proceeds smoothly without formation of insoluble aluminumsalts which hinder the reaction. Following the reduction or hydrolysisstep, the R cations are replaced with hydrogen by means known in theart, for example by acidification and extraction of the acid compoundinto an organic phase.

The desired Formula-XII hydroxy products are isolated by filtration ofthe reaction mixture, advantageously after addition of magnesium sulfateas a filter aid, and evaporation of the organic diluents. TheFormula-XII products are then hydrolyzed if desired to remove Si(A) fromC-l5, and the Hot and 113 products of Formula XIII are separated, ifdesired, by procedures known in the art, e.g., chromatography on silicagel.

The products of Formula-XII are all of the PGE -type and include PGE PGEIS-acetate, I GE methyl ester, PGE IS-acetate methyl ester, PGE and PGEmethyl ester with an -O-Si(A) at C-l 5, the corresponding 15B compounds,and compounds corresponding to all of those wherein hydroxy is attachedto C-Il in beta configuration.

As mentioned above, the transformation of X to XI to Xll usually gives amixture of Formula-XII PGE-type products, part with alpha and part withbeta configuration for the hydroxy at C-1 1. There are severalalternatives regarding that mixture. If O-Si(A) is at tached to C-15,that can readily be transformed by hydrolysis to OI-l. These hydrolysesare carried out by prior art procedures known to be useful fortransforming silyl ethers to alcohols. See, for example, Pierce, citedabove, especially p. 447 thereof. A mixture of water and sufficient of awater-miscible organic diluent to give a homogeneous hydrolysis reactionmixture represents a suitable reaction medium. Addition of a catalyticamount of an organic or inorganic acid hastens the hydrolysis. Thelength of time required for the hydrolysis is determined in part by thehydrolysis temperature. With a mixture of water and methanol at 25 C.,several hours is usually sufficient for hydrolysis. At 0 C., severaldays is usually necessary. Also, if OCOCI-I is attached to C-l5, thatcan readily be transformed to -OI-l by acid-catalyzed alcoholysis asdescribed above for removing the acetyl group of the Formula-VIII and-IX PGA-type starting materials. Both of those transformations are shownin Chart A, i.e., XII to XIII. Either before or after thosetransformations of XII to XIII, the Formula-XII or -Xlll mixture of Ilaand [I3 isomers can be separated by methods known in the art,advantageously by chromatography on silica gel.

Further regarding the Formula-XIII compounds, those compounds whereinthe configuration of the hydroxy at (L11 is beta are within the scope ofthe Formula-V novel compounds of this invention, and those compoundswherein the configuration of the hydroxy at C-ll is alpha and B is arewithin the scope of the Formula-VII novel compounds of this invention.Both groups of novel compounds are used for pharmacological purposesdescribed above for those compounds, the acids also being useful asreactants to prepare pharmacologically useful esters andpharmacologically acceptable and useful salts, both as described above.Moreover, Formula-XIII compounds wherein the configuration of thehydroxy at C-ll is alpha and B is are PGE and PGE methyl ester, both ofknown pharmacological utility.

Still further regarding the separated compounds of Formulas XII andXIII, when a compound with one configuration at C-1 1, either alpha orbeta, is desired as an intermediate or for pharmacological purposes, theother isomer is readily dehydrated to give additional Formula-X PGA-typestarting material which is then used as a starting material according tothe processes defined in Chart A and procedures described above to giveadditional of the desired isomer. These dehydrations are accomplished byprocedures known in the art for dehydration of PGE-type compounds toPGA-type compounds. See, for example, Pike, et al., Proc. NobelSymposium 1], Stockholm (1966),Interscience Publishers, New York, p. 162(1967), and British Specification No. l.097,533. These are acidicdehydrations, and alkanoic acids of 2 to 6 carbon atoms, inclusive,especially acetic acid, are preferred for this purpose. Dilute aqueoussolutions of mineral acid, e.g., hydrochloric acid, especially in thepresence of a solubilizing diluent, e.g., tetrahydrofuran, are alsouseful as reagents for these acidic dehydrations, although thesereagents may also cause partial hydrolysis of the Formula-XII or -XIVmethyl esters to carboxylic acids. ASi(A) moiety at C-l is also removedduring all of these acidic dehydrations.

Still further regarding the Formula-XII and -Xlll compounds, either asmixtures or separately, any of those is transformed to other usefulcompounds or mixtures by changing these POE-type compounds to PGF- typeproducts by reducing the ring carbonyl at C-9 to alpha hydroxy or betahydroxy. Those transformations are shown in Chart B.

In Chart B, R, is hydrogen, methyl, or -Si(A) R is hydrogen or methyl, Ris hydrogen or Si(A) and G is I wherein R is hydrogen or Si(A) when R,and R are hydrogen; R is hydrogen, acetyl, or Si(A) when R and R aremethyl; and R is Si(A when R, is Si-(A) wherein A is as defined above,with the proviso that when R is Si(A) R is also Si- (A) Further in ChartB, B in Formula XVI is H OH,

and indicates attachment to the cyclopentane ring in alpha or betaposition.

The Chart B starting material Xll is prepared as shown in Chart A. Thecompounds of Formula Xlll in Chart A are included in Formula XII. Asdescribed above, 15B-PGE CHART B 'WOORZ XI l HQ (5i lylation XIV(reduction (hydrolysis or a lcoholys is "\zz/ E xv H ISB-PGE acetate,l5[3PGE methyl ester. and ISB-PGE methyl ester acetate are obtained fromPlexaura homomalla (Esper), I792, forma R. All of those compounds areencompassed by Formula XII. and thus, extraction of this form ofPlexaura homomalla provides an alternative source of these startingmaterials.

Referring to Chart B, the starting material XII can be a mixture ofcompounds with regard to the configuration of C-Il, or the startingmaterial can be sterochemically pure with respect to C-l 1, dependingupon whether there has been an earlier separation of Ila and IIB isomers(see above discussion of Chart A reactions).

The transformation of PGE-type starting material XII to PGF-type productXVI involves reduction of a ring carbonyl to a ring hydroxy. Thisprocess is known in the art for some of the compounds encompassed byFormula XII, i.e., when the configuration at C-II is alpha and theconfiguration at C- is S. For the other compounds encompassed by FormulaXII, this reaction is novel, and novel Formula-XV and XVI compounds areproduced.

For this carbonyl-to-hydroxy reduction, methods known in the art areused. See, for example, Pike el al., J. Org. Chem. 34, 3552 (1969). Useis made of any of the known ketonic carbonyl reducing agents which donot reduce ester or acid groups or carbon-carbon double bonds. Examplesof those are the metal borohydrides, especially sodium, potassium,lithium, and zinc borohydrides, lithium (tri-tert-butoxy) aluminumhydride, metal trialkoxy borohydrides, e.g., sodiumtrimethoxyborohydride, and diisobutylaluminum hydride. The sodium,potassium, and zinc borohydrides are preferred for this reduction,especially zinc borohydride.

Unexpectedly, the amalgamated aluminum metal found useful above intransforming the Formula-XI epoxides to Formula-XII hydroxy compoundshas also been found useful as an agent for this carbonyl-tohydroxyreduction of PGE-typ'e compounds to PGF- type compounds. Either the PGE-type salts or the PGE- type esters are employed, for example theFormula-XII hydroxy compounds produced from the Formula-XI epoxides withor without intermediate isolation. Furthermore, the Formula-XI epoxidesmay be subjected to the combined epoxide-reduction and carbonylreductionreactions practically simultaneously by operating at highertemperatures, for example 40-60 C., although it is preferred for highyields of the 11- hydroxy compounds that the reductions be donestepwise. The solvents which are operable for this reduction aregenerally the same as those found useful for the epoxide-reduction step.Somewhat higher temperatures or longer reaction times are required forthe carbonyl-to-hydroxy reduction, however. For example, at 25C., about4 to 24 hours are required; at higher temperatures, e.g., 5060 C., aboutI to 2 hours are sufficient.

This carbonyl reduction usually produces a mixture of PGF a type and PGF3 type compounds, i.e., compounds with the alpha configuration andcompounds with the beta configuration for the hydroxy at C-9. Thismixture of alpha and beta somers is separated by methods known in theart, e.g., chromatography on silica gel. See Pike, et al., ibid., forexample. If the Formula-XII starting material is a mixture of Ila andIIB isomers, then this reduction will usually produce four isomers,i.e., 9a, Ila, 901, 11B, 96, Ila, and 9B, 113. Those compounds are alsoseparated from such mixtures by silica gel chromatography.

Regarding the transformation of XII to XIV in Chart B, it will beobserved that the parameters for XII are such that all XIV compounds areincluded in XIV. In other words, the transformation XII to XIV is anoptional process step in proceeding from XII to XV. The reason for thisis as follows. During the reduction of XIV to XV, the ratio ofJot-hydroxy and QB-hydroxy compounds formed will be different when R, inXIV is hydrogen than when R, is Si(A) For example. with the Formula-XIVcompound wherein R is hydrogen, G is and R O- represents HO i.e.,Ila-hydroxy, sodium borohydride reduction gives 42 parts of thecorresponding Formula-XV 9a-hydroxy compound, and 58 parts of the9B-hydroxy compound. But with the Formula-XIV compound wherein R ishydrogen, G is XV C9 isomer desired and the influence of silylation onthe isomer ratio. For any particular Formula-XIV starting material, thelatter is readily determined by small scale reduction with and withoutsilylation. When silylation before carbonyl reduction is indicated,largely for economic reasons, it is preferred that A be methyl, i.e.,that R, be (CH Si.

This transformation of XII to XIV wherein R is Si- -(A) is carried outas described above for the transformation of hydroxy to --OSi(A) at C-l5prior to the Chart A reactions. When R in XII is hydrogen, the COOH isalso transformed in part or entirely to COOSi-(A) with prolongedsilylation and excess silylating agent. It is optional in transformingXII to XIV wherein R is Si(A) whether or not the COOH of XII isesterified to COOSi(A) When G in Formula XIV is those OH are alsotransformed to C-Si(A) by this silylation.

With regard to the Formula-XV carbonyl reduction product (Chart B), whenthe method used to isolate said product does not remove any Si(A) groupswhich may be present, that is accomplished as described above for theremoval of Si (A)3 groups from Formula-XII products (Chart A, XII toXIII). Also, when G in Formula XV is H OCOCHQ fi OCOCH the acetyl isremoved by alcoholysis also as described above for changing acetoxy atC-I5 to hydroxy. These reactions are shown in Chart B as XV to XVI.

When R in Formula XVI is methyl and the compound wherein R is hydrogenis desired, that methyl ester is saponified, by methods known in theart. See, for example, Just, et al., I. Am. Chem. Soc. 91, 5,371 (1969).This saponification also changes a C-15 acetate to a Cl hydroxy.

The compounds encompassed by Formula XVI include the known compounds PGFPGF B and the methyl esters of those. Also included in Formula XVI arethe novel compounds ISB-PGF a I5 PGF and the methyl esters of those. Allof these new and old compounds are -hydroxy compounds. Also included inFormula XVI are the corresponding but novel llfi-hydroxy compounds whichare also encompassed by Formula V and which are useful for thepharmacological purposes described above either as such or transformedinto salts or esters as described above.

When one of these Formula-XVI compounds has the R or epi configurationfor the hydroxy at Cl5, and the corresponding compound with the Sconfiguration at Cl5 is desired, or when one of these Formula-XVIcompounds has the S configuration for the hydroxy at C-15, and thecorresponding compound with the R or epi configuration at C1 5 isdesired, those desired compounds are made by the processes of Chart C.In Chart C, R R R B, and are as defined above.

The overall process scheme ofChart C is to start with one particular Cl5isomer of a compound encompassed by Formula XVI, i.e., either (8) or15(R). The Cl5 hydroxy of that isomer is oxidized to a ketonic carbonyl(XVII). Then, after an optional silylation of the C-9 and C-1 lhydroxygroups (XVIII), the C-1 5 carbonyl is reduced back to a secondaryhydroxy group. That reduction produces two Cl5 hydroxy isomers, one withS configuration and one with R or epi configuration. After removal ofany silyl groups, the isomers XIX and XX are separated. One of theisomers will be the same compound used as starting material (XVI). Theother isomer will be the desired product. The starting material isomeris recycled to produce more of the desired isomer. This reaction schemehas previously been used to transform PGF, a to 15BPGF See Pike, et al.,J. Org. Chem. 34, 3,552 (1969).

Referring now to Chart C, starting material XVI (from (si l ylation)Chart B) is a single compound, a mixture of two compounds, one withalpha and one with beta configuration at C9, or a mixture of fourcompounds, i.e., 901,1 la, 901,118, 98,1111, and 913,1 113.

For the oxidation of XVI to XVII, any oxidizing agent can be used whichwill oxidize an allylic alcohol to an a,B-unsaturated ketone oraldehyde. Examples of those are2,3-dichloro-5,6-dicyano-l,4-benzoquinone. activated manganese dioxide,or nickel peroxide (see Fieser, et al., Reagents for Organic Syntheses,John Wiley & Sons, Inc., New York, N.Y., 1967, pp. 215, 637, and 731).Alternatively, these oxidations are carried out by oxygenation in thepresence of the 15-hydroxyprostaglandin dehydrogenase of swine lung (seeArkiv for Kemi 25, 293 (1966)). These reagents are used according toprocedures known in the art. See. for example, J. Biol. Chem. 239, 4097(1964).

Regarding the transformation of XVII to XVIII in Chart C, it will beobserved that the parameters for XVIII are such that all XVII compoundsare included in XVIII. In other words, the transformation of XVII toXVIII is an optional process step in proceeding from XVII to XIX and XX.The reason for this is as follows. During the reduction of XVIII to XIXand XX, the ratio of XIX to XX obtained will be different when R, inXVIII is hydrogen than when R-, is Si(A) For example, reduction of theFormula-XVIII 901,1 lat-isomer wherein R and R are both hydrogen withzinc borohydride gives the corresponding Formula-XIX and -)(X in theamounts of 43 parts of XIX (R or epi configuration) and 57 parts of XX(S configuration). On the other hand, when R, in the Formula-XVIIIreactant is Si(A) the amounts with the same reducing agent are 27 partsof XIX and 73 parts of XX. Similar differences are observed with theother isomers encompassed by Formula XVII although not necessarily inthe same direction. Accordingly, whether R in formula XVIII is to behydrogen or -Si(A) depends on the particular Cl5 isomer desired and theinfluence of silylation on the isomer ratio. For any particularFormula-XVII starting material, the latter is readily determined bysmall scale reductions with and without silylation. When silylationbefore carbonyl reduction 18 indi- 29 30 cated, largely for economicreasons, it is preferred that S Z 5- ta ta methyl ester A be methyl,i.e., that R-, be (CH Si. 1 These silylations are carried out asdescribed above y for the Chart A and Chart B silylation. 15B-PGE2 11-Si-(A)3 ether l5-a methyl ester The carbonyl reduction of XVlIl to XIX iscarried 5 out as described above for the transformation of PGE- typeFormula-XIV compounds to PGF-type Formula- 155'PGF2a and 155'PGF2el5'acetate "Ethyl e518 XV compounds. As for those reductions, thesodium, potassium and zinc borohydrides are preferred as re- (reduction,hydrolysis) (separation) ducing agents, especially zinc borohydride. l156"PGF2fl l'acctate methyl ester When the method used to isolate thecarbonyl reduci (saponi fi cation) tion product does not remove anySi-(A) groups which may be present, that is accomplished as del5B'PGF2scribed above for the removal of Si(A) groups 1 (oxidation) fromFormula-XII products (Chart A, XII to XIII). 15

The Formula-XIX and -XX products are separated from each other bymethods known in the art, for ex- L(si ly lation) ample, silica gelchromatography. See, for example, Pike, et al., J. Org. Chem. 34, 3552(1969) for this type Of separation. l (reduction, hydrolysis) If one ofthe isomers or isomer mixtures of Formulas XIX or XX is not desired fora pharmacological use as such or transformed to esters orpharmacologically ac- Fa rat r l\ ce table salts as described above,that isomer or isomer m i i tture is recycled as a Formula-XVI startingmaterial 15B PGFZ QL PGFza in the processes ofChart C to produceadditional of the desired isomer.

The products of Formulas XIX and XX wherein the configuration of theC-11 hydroxy is beta are encom- CHART E passed by Formula V. Theproducts of Formula XIX wherein the configuration of the C-ll hydroxy isalpha are encompassed by Formula VI. The intermediates of Formula XVIIare encompassed by Formula VII. Thus, all of the compounds are usefulfor the pharmacolog- Q ical purposes described above for the Formula V,VI and VII compounds. The compounds prepared as in PGAZ 15 Si (A)3 otherChart C are also useful to make the other esters and thepharmacologically acceptable salts of the Formula V, VI, and VIIcompounds also as described above. (Oxidation) There are two particularembodiments of the novel process of this invention which are especiallypreferred. One of those embodiments provides an optional PGAZ 15-si (A)-ether 0. and '3 lollbepoxides route to PGF a and starts with l5B-PGAacetate methyl ester, the most abundant component of Plexaura homomalla(Esper), 1,792, forma R. The other (reduction, hydrolysis) embodimentprovides a preferred route of PGE and starts with PGA readily obtainedas described above by maintaining coloniesor colony pieces of PlexauraPGE2 and llB-PGE homomalla (Esper), 1,792, forma S in contact with waterin a temperature range up to 50 C. until substantially free of PGA I5acetate methyl ester. (separation) The first of these embodiments isshown in Chart D, and the second is shown in Chart E. All of these ChartV D and Chart E reactions and reagents for effecting S5 G them are15-0x0- PGFBQ 9,1l-di -Si (Al ethcr lSB-PGF and G zo,

PGA

(s lylation) gum 0 described generically and specifically above, and allare exemplified below. In Charts D and E, it is preferred that -Si(A) be-Si(CH Also in Charts 1B-PGA thlet a as 5 2 acetate me y (5 er D and E,it is optional whether silylation of l5-oxol PGF (Chart D) or PGA (ChartE) produces the 15B-PGA2 ace ta te methyl ester (1 and f 10, ll-epoxides Corresponding Sl (A)3 ester'elher or y thfi ether- The invention ismore fully understood by the follow- 7 ing Examples and Preparations:15B-PGE2 and 116,15B-PGE l5-acetate methyl esters All temperatures arein degrees centigrade.

Ultraviolet spectra are recorded on a Cary Model 15 l (separatton)spectrophotometer.

(reduction) The collection of chromatographic eluate fractions startswhen the eluant front reaches the bottom of the column.

Brine, herein, refers to an aqueous saturated sodium chloride solution.

The A-lX solvent system used in thin layer chromatography (TLC) is madeup from ethyl acetate-acetic acid-2,2,4-trimethylpentane-water(90:20:501100) according to M. l-lamberg and B. Samuelsson, J. Biol.Chem. 241, 257 (1966).

Preparation 1 To distinguish Plexaura homomalla (Esper), 1,792, forma Rfrom Plexaura homomalla (Esper), 1,792, forma S, a TLC method is used. Aspecimen approximately 2 cm. in length is harvested and placed in asmall vial, with a small amount of water if necessary to insure it iswet, and kept closed for 6-24 hrs. About one ml. of methanol is thenadded and the sample is either shaken for 2 hrs. at about 25 C. or isstored for 16-24 hrs. at about 10 C. A sample of the liquid (10-21 A) isspotted on a TLC plate. It is preferred to use a fluorescent-treatedsilica gel plate, e.g., Uniplate Silica Gel GF (Analtech, lne., Newark,Del.). As reference standards, spots of PGA and BPGA are also applied.The plate is developed in the A-IX system (Hamberg and Samuelsson, J.Biol. Chem. 241, 257 (1965)). The spots are finally visualized withvanillin-phosphoric acid spray (McAleer, Arch. Biochem. E. Biophys. 66,120 1957)). Comparison ofthe unknown with the two reference spots isthen made and the identity of the coral established (forma Scorresponding to PGA forma R to 15B-PGA Preparation 2 PGA from Plexaurahomomalla (Esper), forma S.

Colonies of Plexaura homomalla (Esper), 1,792, forma S, collected fromreefs off the north shore of Jamaica, are frozen by contact with solidcarbon dioxide within 1 hour after removal from the reef waters. Thefrozen colonies are maintained in insulated boxes containing solidcarbon dioxide (temperature below about C.) until ready for thawing.Then, the frozen colonies (700 g.) are ground to a small particle size(Waring blender) and mixed with 1,500 ml. of water. The mixture ismaintained about 20 hrs. at about C. with stirring. Then, the mixture isfiltered through a pad of diatomaceous earth, and the filtrate isacidified with concentrated hydrochloric acid to pH about 2-3. Theacidified filtrate is extracted four times with ethyl acetate. Theextracts are combined, filtered, washed with brine, dried with anhydroussodium sulfate, and evaporated under reduced pressure to give 1 1 g. ofoily residue.

The solid residue on the diatomaceous earth filter pad is stirred 2hours in methanol (enough to cover said residue) at 25 C. The mixture isthen filtered, and the filtrate is evaporated to give 14 g. of oilyresidue.

The two oily residues are combined and chromatographed on 1,500 g. ofacid-washed silica gel, eluting successively with 8 l.k ofa 25 to 65percent gradient of ethyl acetate in Skellysolve B, 8 1. of a 65 to 100percent gradient of ethyl acetate in Skellysolve B, and 5 l. of 2percent methanol in ethyl acetate, collecting 500 ml. fractions.(Skellysolve B is a mixture of isomeric hexanes). Fractions 8-12 arecombined and evaporated to give a small amount of PGA containing a traceof PGA methyl ester. Fractions 15-18 are combined and evaporated to give9.54 g. of PGA Fractions 35-40 are combined to give 0.414 g. of PGEPreparation 3 l5B-PGA from Plexaura Homomalla 1,792, forma R.

Colonies of Plexaura homomalla (Esper), 1,792, forma R, collected fromreefs off the southeast shore of Florida near Miami, are chopped intochunks several inches long. The chunks are frozen by contact with solidcarbon dioxide with 1 hour after removal from the reef waters. Thefrozen colony pieces are maintained in insulated boxes containing solidcarbon dioxide (temperature below about -20 C.) until ready for thawing.Then, colony pieces (600 g.) are mixed with 1.500 ml. of water. Themixture is stirred and maintained at 25 C. for 23 hours. The mixture isthen filtered through a pad of diatomaceous earth, and the filtrate isacidified to pH about 2-3 with concentrated hydrochloric acid. Theacidified filtrate is extracted four times with ethyl acetate. Theextracts are combined, filtered, washed with brine, dried with anhydroussodium sultate, and evaporated to give 9.2 g. of oily residue.

The solid residue on the diatomaceous earth pad is stirred 15 hours inmethanol (enough to cover said residue) at 25 C. The mixture is thenfiltered, and the filtrate is evaporated. The residue is dissolved inethyl acetate, and the solution washed successively with 3 Nhydrochloric acid and brine, dried with anhydrous sodium sulfate, andevaporated to give 5.83 g. of an oily residue.

The second oily residue and 8.2 g. of the first oily residue arecombined and chromatographed on one kg. of acid-washed silica gel,eluting successively with 3-1. portions of 25, 35, 45, 55, and 65percent ethyl acetate in Skellysolve B, collecting 500-ml. fractions.Fractions 18-22 are combined and evaporated to give 5.54 g. of l5BPGAFractions 15-17 are combined and evaporated to give 1.37 g. of ISB-PGAmethyl ester.

Preparation 4 PGA compounds (Esper), 1792, forma S.

Frozen colonies of Plexaura homomalla (Esper), 1,792, forma S (seePreparation 2) are broken manually into pieces several cm. in length.The pieces (500 g.) are then covered with methanol and the mixture ismaintained for 3 hours at 25 C. The mixture is then ground in a Waringblender and filtered, and the filtrate is evaporated under reducedpressure. The residue is dissolved in ethyl acetate, and the solution iswashed successively with one N hydrochloric aicd, water, and brine,dried with anhydrous sodium sulfate, and evaporated under reducedpressure. The oily residue is chromatographed on 2 kg. of acid-washedsilica gel wetpacked with Skellysolve B (a mixture of isomeric hexanes),eluting with 241. ofa 25 to percent ethyl acetate in Skellysolve Bgradient. The fractions which contain PGA acetate methyl ester, PGAacetate, PGA methyl ester, and PGA as shown by TLC with the A-IX systemare separately combined and evaporated to give those compounds.

Preparation 5 15B-PGA compounds from Plexaura homomalla (Esper), 1792,forma R.

Colonies of Plexaura homomalla (Esper), 1,792, forma R, collected fromreefs off the southeast shore of Florida near Miami, are chopped intochunks several inches long. The chunks are frozen by contact with(Esper),

from Plexaura homomalla solid carbon dioxide within 1 hour after renovalfrom the reef waters. The frozen colony pieces are maintained ininsulated boxes containing solid carbon dioxide (temperature below about20 C.) until the time for extraction. Then, the frozen colony pieces areground to a small particle size (Mitts and Merrill hogger; averagelargest dimension about mm). The particles (1,500 g.) are then stirredat high speed with 5 gallons of dichloromethane for 20 minutes at about25 C. external temperature. The mixture of dichloromethane and particlesis then filtered through a pad of diatomaceous earth, and the filtrateis evaporated to about a 2- liter volume at 30 C. under reducedpressure. The liquid which remains is washed with waer, dried withsodium sulfate, and evaporated at 30 C. under reduced pressure.

The oily residue (60 g.) is ohromatographed on 3 kg. of silica gel wetpacked in Skellysolve B (a mixture of isomeric hexanes), elutingsuccessively with a gradient of4 l. of Skellysolve B and 4 l. of 20percent ethyl acetate in Skellysolve B, 27 l. of 20 percent, 18 l. of 50percent and 8 l. of 75 percent ethyl acetate in Skellysolve B,collecting 600-ml. fractions. Fractions 39-60 are combined andevaporated to give 24.3 g. of 15BP- GA acetate methyl ester. Betweenfractions 60 and 74 those fractions shown by TLC to contain 15BPGAacetate are combined and evaporated to yield that compound. Fractions74-76 are combined and evaporated to give 1.03 g. of 15[3PGA methylester. Fractions 83-91 are combined and evaporated to give 1.08 g. of15BPGE 15-acetate methyl ester. Still later fractions shown by TLC tocontain 15BPGE methyl ester are combined and evaporated to yield thatcompound.

Detection of the respective compounds by TLC is done by methods known inthe art, e.g., by spotting the extract fractions on a TLC silica gelplate alongside spots of the authentic compounds, developing the platewith the A-IX system, and observing which spots of the extract fractionscorrespond exactly to the spots of the authentic compounds.

Following the procedures of Preparation 5, but substituting Plexaurahomomalla (Esper), 1,792, forma S for the Plexaura homomalla (Esper),1,792, forma R of that example, there are obtained the correspondingcompounds of 15(S) configuration, viz.: PGA acetate methyl ester, PGAacetate, PGA methyl ester, PGE IS-acetate methyl ester, and PGE methylester.

Preparation 6 PGA and 5,6-trans-PGA Separation of PGA from 5,6-trans-PGAis done on a chromatographic column using a silver-saturated ionexchangeresin. Preferably a macroreticular ion exchange resin is used, e.g., asulfonated styrene-d vinylbenzene copolymer having surface area of 40-50sq. m./g., 30-40 percent porosity, and total exchange capacity of4.5-5.0 meq. per gram of dry resin, for example Amberlyst 15, availablefrom Rohm and Haas Co., Philadelphia, Pa. The acid-form resin is packedin a column, washed with warm water, and converted to the silver form bypassing a 10 percent silver nitrate solution through the column untilthe effluent shows a pH of 3.5-4.0. The column is then washed with waterto remove ionic silver, and finally with denaturated ethanol (Type 3A).A solution of a mixture of PGA, and 5,6-trans-PGA e.g., fractions -18 ofPreparation 2, in ethanol is charged to the column. Elution with 3Aalcohol then yields fractions which are combined according to theircontent of 5,6-trans-PGA (fastereluting) or PGA Testing for the presenceof 5,6-trans- PGA or PGA in the eluate is conveniently done by TLC usingsilver nitrate-treated silica gel plates (e.g., Analtech Uniplatesdipped in saturated ethanolic silver nitrate and dried) and developingwith the A-lX system. R, of PGA is 0.45; R, of 5,6-trans-PGA is 0.50.Combined fractions are concentrated, partitioned between dichloromethaneand a little water, dried over sodium sulfate, and concentrated underreduced pressure to yield the title compounds.

For quantitatively assaying the 5,6-trans-PGA content of mixtures of PGAand 5,6-trans-PGA a combinationthin-layer-spectrophotometric assay isused. Silica gel-impregnated glass microfiber sheets (e.g., lTLC sheetsof the Gelman Instrument Co., Ann Arbor, Mich.) are impregnated withsilver nitrate, using 5 percent ethanolic silver nitrate and drying.Spots of 100 to 200 ,ug of the PGA mixture are applied and developed inthe solvent system 2,2,4-trimethylpentane: ethyl acetate: acetic acid:water (100:35z8210, upper phase). The sheet is dried and sprayed withRhodamine 6G (Applied Science Co., State College, Pa) and viewed underultraviolet light. The areas containing the cis and trans materials (R,of PGA 0.6; R, of 5,6-trans- PGA 0.7) are marked, then excised andeluted with methanol (1.9 ml.) and potassium hydroxide solution (0.1 ml.of 45 percent). After incubation at 40 for 30 min., the respectivesolutions are centrifuged and analyzed spectrophotometrically at 278 nm.

Following the procedure of Preparation 6, 5,6-trans- 15BPGA is separatedfrom 15BPGA Preparation 7 PGE and 5,6-trans-PGE Following the procedureof Preparation 6, PGE is separated from 5 ,6-trans-PGE as follows: Asolution of a mixture of PGE and 5,6-trans-PGE is charged to the column.Elution with 3A alcohol yields fractions which are combined according totheir content of 5,6- trans-PGE (faster eluting) or PGE Assay for 5,6-trans-PGE or PGE is done by TLC as for the PGA type compounds above. R,of PGE is 0.13; R, of 5,6- trans-PGE is 0.17. Combined fractions areconcentrated, dried over sodium sulfate, and concentrated under reducedpressure to yield the title compounds.

Preparation 8 PGA 15-Acetate Methyl Ester, separation from 5,6-Trans-PGA l5-Acetate Methyl Ester.

A mixture of PGA l5-acetate methyl ester and 5,6- trans-PGA 15-acetatemethyl ester (11.0 g., :15) is dissolved in 415 ml. of a solution ofmethanol-wateracetic acid (-5-04) and mercuric acetate (6.1 g.), andleft standing at about 25 C. for 30 min. Water (250 ml.) is added andthe mixture extracted twice with 700 ml. of Skellysolve B. TheSkellysolve B phase is washed with ml. of 60 percent methanol, driedover sodium sulfate, and concentrated to an oil (4.35 g.) having a highcontent of 5,6-trans-PGA l5-acetate methyl ester. The aqueous methanolphase is acidified with 32 ml. of 6 N. hydrochloric acid and the mixtureis extracted with two portions of 700 ml. of Skellysolve B. The organicphase is dried over sodium sulfate and concentrated to an oil (5.53 g.)This last material is subjected to the same procedures again, using 350ml. of the methanol-water-acetic acid and 4.6 g. of mercuric acetate.There is recovered from the work-up of the aqueousmethanol phase afraction (3.92 g.) of the title compound containing only a smallpercentage of the 5,6-trans-PGA compound.

Following the procedure of Preparation 8, 5,6-trans- 15BPGA lS-acetatemethyl ester is separated from 15B-PGH IS-acetate methyl ester.

In the following examples, the above-described 5,6- trans-PG and5,6-trans15BPG compounds are subjected to the same transformations asthe P6 and 15B-PG compounds disclosed herein and illustrated hereafter.

Example 1 ISB-PGA Methyl Ester.

A solution of 70 percent aqueous perchloric acid (50 drops) in 50 dropsof water is added to a solution of 15BPGA acetate methyl ester (2.0 g.)in 100 ml. of methanol. The mixture is stirred for 15 hours at 25 C. andthen diluted with 80 ml. of water. The methanol is removed under reducedpressure, and the aqueous residue is extracted with ethyl acetate. Theextract is washed successively with water and brine, dried withanhydrous sodium sulfate, and evaporated. The residue is chromatographedon 200 g. of silica gel, eluting with 2.5 l. of a gradient of 20-70percent ethyl acetate in Skellysolve B (a mixture of isomeric hexanes),collecting 100-ml. fractions. Fractions 15-19 are combined andevaporated to give 727 mg. of 15BPGA methyl ester.

Example 2 PGA Methyl Ester.

A solution of 15B-PGA methyl ester (250 mg.) in 20 ml. of anhydroustetrahydrofuran is cooled to C. in an atmosphere of nitrogen.Tributylamine (1.5 ml.) is added, and the mixture is stirred at 0 C.while adding methanesulfonyl chloride (1 ml.) dropwise. The mixture isstirred 30 minutes at 0 C. Then, 10 ml. of water is added, and themixture is allowed to warm to 25 C. and is stirred for 1 hour. Thetetrahydrofuran is evaporated under reduced pressure, and the aqueousresidue is extracted with ethyl acetate. The extract is washedsuccessively with l N hydrochloric acid, water, and brine, dried withanhydrous sodium sulfate, and evaporated. The residue is chromatographedon 30 g. ofsilica gel, eluting with 800 ml. ofa gradient of 20-70percent ethyl acetate in Skellysolve B, collecting 25-ml. fractions.Fractions 14-16 are combined and evaporated to give 58 mg. of PGA;.methyl ester. Fractions 12 and 13 are combined to give 49 mg. of thestarting material, 15B-PGA methyl ester.

Following the procedure of Example 2, PGA methyl ester is transformed toa mixture of PGA and 15B-- PGA methyl esters, the two compounds beingseparated as in Example 2.

Example 3 PGA l5-formate and 15BPGA 15-formate.

A solution of sodium carbonate (50 mg.) in 7.5 ml. of anhydrous formicacid is added to PGA, (0.25 g.). This mixture is stirred under nitrogenat 25 C. for 2 hrs. The reaction mixture is concentrated under reducedpressure, taken up in'benzene, and again concentrated under reducedpressure. The residue is chromatographed on acid-washed silica gel(e.g., Mallinckrodt Silicar CC-4), eluting with a gradient of 25-75percent ethyl acetate-Skellysolve B (isomeric hexane mixture) andcollecting fractions. Those fractions shown by TLC to contain therespective IS-f'ormate compound, separated from its isomer and free ofstarting material and impurities, are combined and concentrated underreduced pressure to give the title compounds.

Example 4 PGA and 15BPGA PGA l5-formate (100 mg., Example 3) isdissolved in a mixture of 10 ml. of methanol and 2.5 ml. of saturatedaqueous sodium bicarbonate solution. The solution is stirred undernitrogen at 25 C. for 2.5 hrs. Then 5 ml. of water and 2 ml. of l N.hydrochloric acid are added, and the solution concentrated. The aqueousresidue is adjusted to pH 2-3 and extracted three times with ethylacetate. The combined extracts are washed with water, dried over sodiumsulfate, and concentrated to yield PGA Similarly, hydrolysis of l5B-PGAl5-formate (Example 3) yields l5BPGA Example 5 l5B--PGA 10,11-EpoxideAcetate Methyl Ester.

Hydrogen peroxide (350 ml. 30 percent aqueous) is added with stirring toa solution of l5B---PGA acetate methyl ester (265 g.) in 5.000 ml. ofmethanol under a nitrogen atmosphere at 20 C. Then, one N aqueouspotassium hydroxide solution (50 ml.) is added gradually during 1 hourwith stirring at 20 C. The mixture is stirred an additional 2 hours at-20 C. Then. one N hydrochloric acid ml.) is added, and the methanol isremoved under reduced pressure at 35 C. The residue is dissolved in3,000 ml. of ethyl acetate, and the solution is washed 3 times with500-ml. portions of water. The combined water washes are extracted with300 ml. of ethyl acetate. The two ethyl acetate solutions are combined,washed with brine, dried with anhydrous sodium sulfate and evaporated togive 2.75 g. of a mixture of the alpha and beta 10,1 l-epoxides ofl5B-PGA acetate methyl ester.

Example 6 PGA 10,1 l-Epoxide Methyl Ester.

Hydrogen peroxide (0.3 ml.; 30 percent aqueous) and one N aqueous sodiumhydroxide (0.5 ml.) are added to a solution of PGA methyl ester (229mg.) in 10 ml. of isopropyl alcohol at 0 C. After 2.5 hours at 0 C., 10ml. of water and one ml. of one N hydrochloric acid are added, and theisopropyl alcohol is removed under reduced pressure. The residue isextracted with ethyl acetate. The extract is washed successively withwater and brine, dried with anhydrous sodium sulfate, and evaporated.The residue is chromatographed on 30 g. of silica gel, eluting with 800ml. of a gradient of 20-70 percent ethyl acetate in Skellysolve B,collecting 25-ml. fractions. Fractions 5-10 are combined and evaporatedto give 136 mg. of a mixture of the alpha and beta 10,1l-epoxides ofPGA, methyl ester.

Example 7 PGA Acetate Methyl Ester a and B 10,1 l-Epoxides Refer toChart A.

A solution of PGA l5-acetate methyl ester (1.954 g.) in 30 ml. ofdimethoxyethane (DME) is cooled to 55 C. under nitrogen, and 5.25 ml. oft-butyl hydroperoxide is added. Then, 5 ml. of 0.25 N. methanoliclithium hydroxide (prepared from the mono-hydrate) is added over min.After about 46 hrs. an additional 2.5 ml. of the base is added over 50min. Finally. after about 23.5 hrs. the reaction is complete, as shownby TLC. The mixture is acidified to pH 5-6 with l N. hydrochloric acidand is concentrated under reduced pressure. The residue is taken up inethyl acetate, washed with brine, dried over sodium sulfate, andconcentrated under reduced pressure. The product, 2.0 g., contains thetitle alpha and beta compounds in a ratio of 6:1, respectively, as shownby gas chromatography.

Following the procedures of Example 7, but replacing the lithiumhydroxide solution with methanolic magnesium mcthoxide (prepared frommagnesium and anhydrous methanol), there is obtained a productcontaining the alpha and beta epoxides in a ratio of 4: 1.

Following the procedures of Example 7, but replacing the DME with amixture of toluene-DME (10:1) and replacing the lithium hydroxide withTriton B (benzyltrimethylammonium hydroxide) in methanol, there areobtained the alpha and beta epoxides in a ratio of 7.221.

Following the procedures of Example 7, but replacing the DME with amixture of toluene-DME (1:1) and holding the reaction temperature at 40C., the product contains the alpha and beta epoxides in a ratio of6.2:1.

Following the procedures of Example 7, but replacing the DME withtetrahydrofuran (THF) and replacing the lithium hydroxide solution withTriton B, there are obtained the alpha and beta epoxides in a ratio of4.5:1.

Example 8 15,B--PGE -Acetate Methyl 11B,15/3PGE 15-Acetate Methyl Ester.

Granular aluminum metal (50 g.) is added to a solution of mercuricchloride (50 g.) in 2 l. of water. The mixturue is swirled untilhydrogen gas evolution starts to become vigorous (about 30 seconds).Then, most of the aqueous solution is decanted, and the rest is removedby rapid filtration. The amalgamated aluminum is washed rapidly andsuccessively with two ZOO-m1. portions of methanol and two 200-ml.portions of anhydrous diethyl ether. The amalgamated aluminum is thencovered with anhydrous diethyl ether until used.

Methanol (250 ml.) and water (25 ml.) are added to a solution of amixture of the alpha and beta 10,11-

Ester and epoxides of ISB-PGA acetate methyl ester (275 g.)

in 2,500 ml. of diethyl ether. The mixture is cooled to 10 C. and theamalgamated aluminum prepared as above from 50 g. of aluminum metal isadded. The mixture is stirred and maintained at about 25 C. withexternal cooling. After 1 hour, amalgamated aluminum prepared as abovefrom 50 g. of aluminum metal is added. After an additional hour ofstirring at 25 C., amalgamated aluminum prepared as above from 50 g. ofaluminum metal and also 25 ml. of water are added. After an additionalhour of stirring at 25 C., 100 g. of magnesium sulfate is added as afilter aid, and the mixture is filtered. The filter cake is washedthoroughly with dichloromethane, and the combined filtrate and washingsare evaporated at 25 C. under reduced pressure to give a mixture (247g.) of l5B--PGE 15-acetate methyl ester and 1lB,15BPGE 15-acetate methylester.

Part of this mixture (210 g.) is chromatographed on 30 kg. of silica gelwet-packed with 60 l. of 25 percent ethyl acetate in Skellysolve B(6-inch diameter column), eluting successively with 60-1. portions of25, 30, 35,40, 45, 50, 55, and 60 percent ethyl acetate in SkellysolveB, collecting 4-1. fractions. Fractions 71-76 are combined andevaporated to give 27 g. of llfl,l5B--PGE IS-acetate methyl ester.Fractions 81-98 are combined and evaporated to give g. of ISB-PGEl5-acetate methyl ester.

Example 9 15B-PGE l5-Acetate Methyl 11B,l5BPGE 15-Acetate Methyl Ester.

Anhydrous sodium acetate (0.5 g.) and zinc dust (500 mg.) are added to asolution of a mixture of the alpha and beta epoxides of l5B-PGA acetatemethyl ester, prepared as in Example 5, in 5 ml. of acetic acid. Thismixture is stirred at 25 C. in an atmosphere ofnitrogen and cooled toabout 15 C. One-halfml. ofa solution of chromium (lll) chloridehexahydrute (300 mg.) in 1 ml. of water is added, and the mixture isstirred at 0 C. for 3 hours. The mixture is then diluted with ethylacetate, and the solution is washed successively with four portions ofwater, one N hydrochloric acid, sodium bicarbonate solution, and brine,dried with anhydrous sodium sulfate, and evaporated. The residue ischromatographed on 20 g. of silica gel, eluting with 600 ml. of agradient of 20-75 percent ethyl acetate in Skellysolve B, collecting25-m1. fractions. Fractions 10 and 11 are combined to give 113,153-- PGEl5-acetate methyl ester. Fractions l3 and 14 are combined to give 15BPGE15-acetate methyl ester.

Example 10 PGE Methyl Ester and ll,B-PGE Methyl Ester.

Freshly prepared chromium (ll) acetate (450 mg., argon atmosphere;Inorganic Syntheses, 8, is added to a solution of 136 mg. of epoxides(Example 6) in a mixture of 3 ml. of acetic acid and one ml. of water inan atmosphere of argon at 0 C. The mixture is stirred at 5 C. underargon for 18 hours. Ice is then added to the mixture, and that mixtureis extracted with ethyl acetate. The extract is washed successively withwater, one N hydrochloric acid, sodium bicarbonate solution, and brine,dried with anhydrous sodium sulfate, and evaporated. The residue ischromatographed on silica gel (20 g.), eluting with 600 m1. of agradient of 20-100 percent ethyl acetate in Skellysolve B, collecting 20ml. fractions. Fractions 19-22 are combined and evaporated to give 27mg. of 11B-PGE methyl ester. Fractions 24-27 are combined and evaporatedto give 5 mg. of PGE methyl ester.

Example 11 PGE and llB-PGE Hydrogen peroxide (0.35 ml.; 30 percentaqueous) is added to a solution of PGA (200 mg.) in 5 ml. of methanol.The mixture is cooled to 20 C., and 0.75 ml. of one N aqueous sodiumhydroxide solution is slowly added with stirring. After 1 hour ofstirring at 20 C., one ml. of one N hydrochloric acid is added, and themixture is evaporated under reduced pressure. The residue is dissolvedin ethyl acetate, and the resulting solution is washed successively withwater and brine, dried with anhydrous sodium sulfate, and evaporated.The residue is treated with amalgamated aluminum as described in Example8, using 2.5 ml. diethyl ether, 0.25 ml. methanol, and 0.03 ml. water,the amalgamated aluminum being added in 2 portions. When the reductionis complete, ethyl acetate and one N hydrochloric acid are added to thereaction mixture and the mixture is separated in a separatory funnel.The ethyl acetate layer is washed successively with one N hydrochloricacid, water, and brine, dried with anhydrous sodium sulfate, andevaporated. The residue is subjected to preparative thin layerchromatography to give PGE and llfiL-PGE in the ratio 1:2.

Ester and

2. A compound according to claim 1 wherein the HO * is attached to thering in alpha configuration.
 3. A compound according to claim 2 whereinR1 is hydrogen.
 4. A compound according to claim 1 wherein the HO * isattached to the ring in beta configuration.
 5. A compound according toclaim 4 wherein R1 is hydrogen.